Speaker Abstract RCMM 2017: Associate Professor Dr. Low Teck Yew

A systems-wide screen identifies substrates of the SCF^bTrCP ubiquitin ligase

Teck Yew Low,^1,2 Mao Peng,^1,2* Roberto Magliozzi,^{3 *} Shabaz Mohammed,^1,2† Daniele Guardavaccaro,³ Albert J. R. Heck^1,2‡

Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases. Identification of substrates of ubiquitin ligases is crucial not only to unravel the molecular mechanisms by which the UPS controls protein degradation but also for drug discovery purposes because many established UPS substrates are implicated in disease. We developed a combined bioinformatics and affinity purification– mass spectrometry (AP-MS) workflow for the system-wide identification of substrates of SCF^bTrCP, a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are characterized by a multisubunit architecture typically consisting of the invariable subunits Rbx1, Cul1, and Skp1, and one of 69 F-box proteins. The F-box protein of this member of the family is bTrCP. SCF^bTrCP binds, through the WD40 repeats of bTrCP, to the DpSGXX(X)pS diphosphorylated motif in its substrates. We recovered 27 previously reported SCFb^TrCP substrates, of which 22 were verified by two independent statistical protocols, thereby confirming the reliability of this approach. In addition to known substrates, we identified 221 proteins that contained the DpSGXX(X)pS motif and also interacted specifically with the WD40 repeats of bTrCP. Thus, with SCF^bTrCP, as the example, we showed that integration of structural information, AP-MS, and degron motif mining constitutes an effective method to screen for substrates of ubiquitin ligases.

1. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands.
2. Netherlands Proteomics Center, Padualaan 8, 3584 CH Utrecht, Netherlands.
3. Hubrecht Institute–KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, Netherlands.

*These authors contributed equally to this work.

†Present address: Departments of Chemistry and Biochemistry, University of Oxford, New Biochemistry Building, South Parks Road, Oxford OX1 3QU, UK.